N2 -dansyl-L-arginine derivatives, and the pharmaceutically acceptable acid addition salts thereof

ABSTRACT

N 2  -dansyl-L-arginine esters and amides having the formula ##STR1## or the acid addition salts thereof with a pharmaceutically acceptable acid, wherein R is selected from the group consisting of (1) alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy, respectively, containing not more than 10 carbon atoms, aralkyl of not more than 15 carbon atoms, tetrahydrofurfuryloxy, and alkoxy of not more than 10 carbon atoms substituted with an alkoxy group of not more than 10 carbon atoms, halogen or nitro; (2) ##STR2## wherein R 1  and R 2  are members selected from the group consisting of hydrogen, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, and alkyl, wherein said member has no more than 10 carbon atoms, and wherein said alkyl may be substituted with a member selected from the group consisting of alkoxy, alkoxycarbonyl, arylcarbamoyl, acyl, acyloxy, N,N-polymethylenecarbamoyl, respectively, containing not more than 10 carbon atoms, and carboxyl; and (2) ##STR3## wherein Z is a divalent group containing up to 10 carbon atoms, which consists of more than one group selected from the group consisting of methylene --CH 2 , monosubstituted methylene ##STR4## wherein R 3  is selected from the group consisting of alkyl, acyl, alkoxy, and alkoxycarbonyl, respectively, containing not more than 10 carbon atoms, and carbamoyl, and disubstituted methylene ##STR5## wherein R 4  R 5  are alkyl groups of not more than 10 carbon atoms, and which may further contain at least one member selected from the group consisting of oxy--O--, thio --S--, cycloalkylene of not more than 10 carbon atoms, imino ##STR6## alkyl substituted imino ##STR7## wherein R 6  is an alkyl group of not more than 10 carbon atoms, acyl substituted imino ##STR8## wherein R 7  is an alkyl group of not more than 10 carbon atoms, phenylene ##STR9## and carbonyl ##STR10## which completes the  ring together with the said methylene, mono-substituted methylene or disubstituted methylene.

This is a continuation of application Ser. No. 691,766, filed June 1,1976, now U.S. Pat. No. 4,227,006, which is a division of applicationSer. No. 496,939, filed Aug. 13, 1974, now U.S. Pat. No. 3,978,045.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to certain new and useful N² -dansyl-L-arginineesters and amides, and the pharmaceutically acceptable acid additionsalts thereof, which are of especial value in view of their outstandingantithrombotic properties.

2. Description of the Prior Art

In the past, there have been many attempts to obtain new and improvedagents for the treatment of thrombosis. Of these, N²-(p-tolylsulfonyl)-L-arginine esters are known to be effective indissolving blood clots. (U.S. Pat. No. 3,622,615, patented Nov. 23,1971). A need continues to exist however, for a highly specificinhibitor on thrombin for the control of thrombosis.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a groupof N² -dansyl-L-arginine esters, amides and pharmaceutically acceptableacid addition salts.

Another object of the present invention is to provide a group of N²-dansyl-L-arginine esters, amides and pharmaceutically acceptable acidaddition salts which are useful in the diagnostic selectivedetermination of thrombin in blood and in drug therapy as antithromboticagents.

Briefly, these objects and other objects of the present invention ashereinafter will become more readily apparent can be attained by N²-dansyl-L-arginine esters and amides of formula (I): ##STR11## or theacid addition salts thereof with a pharmaceutically acceptable acid,wherein R is selected from the group consisting of (1) alkoxy,alkenyloxy, alkynyloxy, and cycloalkoxy, respectively, containing notmore than 10 carbon atoms, aralkyl or not more than 15 carbon atoms,tetrahydrofurfuryloxy, and alkoxy of not more than 10 carbon atomssubstituted with an alkoxy group of not more than 10 carbon atoms,halogen or nitro; (2) ##STR12## wherein R₁ and R₂ are members selectedfrom the group consisting of hydrogen, alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl. alkenyl, and alkyl, wherein said member has no morethan 10 carbon atoms, and wherein said alkyl may be substituted with amember selected from the group consisting of alkoxy, alkoxycarbonyl,arylcarbamoyl, acyl, acyloxy, N,N-polymethylenecarbamoyl, respectively,containing not more than 10 carbon atoms, and carboxyl; and (2)##STR13## wherein Z is a divalent group containing up to 10 carbonatoms, which consists of more than one group selected from the groupconsisting of methylene --CH₂, monosubstituted methylene ##STR14##wherein R₃ is selected from the group consisting of alkyl, acyl, alkoxy,and alkoxycarbonyl, respectively, containing not more than 10 carbonatoms, and carbamoyl, and disubstituted methylene ##STR15## wherein R₄and R₅ are alkyl groups of not more than 10 carbon atoms, and which mayfurther contain at least one member selected from the group consistingof oxy--O--, thio--S--, cycloalkylene of not more than 10 carbon atoms,imino ##STR16## alkyl substituted imino ##STR17## wherein R₆ is an alkylgroup of not more than 10 carbon atoms, acyl substituted imino ##STR18##wherein R₇ is an alkyl group of not more than 10 carbon atoms, phenylene##STR19## and carbonyl ##STR20## which completes the ##STR21## ringtogether with the said methylene, monosubstituted methylele ordisubstituted methylene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the formula (I), R is preferably alkoxy of 1-8 carbon atoms,aralkyloxy of 7-9 carbon atoms, alkenyloxy of 3-6 carbon atoms,alkynyloxy of 3-6 carbon atoms, cyclohexyloxy, tetrahydrofurfuryloxy,ω-alkoxyalkyloxy of 2-6 carbon atoms, ω-chloroalkyloxy of 2-6 carbonatoms, 2-nitrobutyloxy, alkylamino of 1-9 carbon atoms,ω-alkoxyalkylamino of 2-6 carbon atoms, ω-alkoxycarbonyl-alkylamino of2-8 carbon atoms, alkenylamino of 3-5 carbon atoms, cyclopropylamino,cyclohexylmethylamino, phenylamino, aralkylamino of 7-10 carbon atoms,2-phenylcarbamoylethylamino, N,N-tetramethylenecarbamoylmethylamino,dialkylamino of 2-10 carbon atoms,N-alkyl-N-(ω-alkoxycarbonylalkyl)amino of 3-7 carbon atoms,N-alkyl-N-aralkylamino of 8-10 carbon atoms,N-alkyl-N-(ω-acylalkyl)amino of 4-8 carbon atoms,N,N-polymethyleneiminyl of 3-10 carbon atoms, N,N-polymethyleneiminyl of3-10 carbon atoms substituted with alkyl of 1-5 carbon atoms,alkoxycarbonyl of 2-5 carbon atoms, acyl of 2-5 carbon atoms, andcarbamoyl; n-(tetrahydro-1,n-oxazinyl), wherein n is the integers 2, 3or 4, n-(tetrahydro-1,n-oxyazinyl), wherein n is the integers 2, 3 or 4and which is substituted by one or two alkyl groups of 1-5 carbon atoms;2-isoindolinyl, 4-alkyl-1-piperazinyl of 5-8 carbon atoms,1,2,3,4-tetrahydro-1-quinolyl, and 4-(4-azabicyclo(3.2.2)nonyl).

Suitable compounds of this invention include N² -dansyl-L-arginineesters and amides such as N² -dansyl-L-arginine ethyl ester, N²-dansyl-L-arginine n-propyl ester, N² -dansyl-L-arginine n-butyl ester,N² -dansyl-L-arginine n-amyl ester, N² -dansyl-L-arginine isopentylester, N² -dansyl-L-arginine n-hexyl ester, N² -dansyl-L-arginine benzylester, N² -dansyl-L-arginine crotyl ester, N² -dansyl-L-arginine3-butynyl ester, N² -dansyl-L-arginine 2-methoxyethyl ester, N²-dansyl-L-arginine 3-chloropropyl ester, N² -dansyl-L-arginine4-chlorobutyl ester, N² -dansyl-N-(n-butyl)-L-argininamide, N²-dansyl-N-(2-methoxyethyl)-L-argininamide, N²-dansyl-N-(2-ethoxyethyl)-L-argininamide, N²-dansyl-N-(2-methoxycarbonylethyl)-L-argininamide, N²-dansyl-N-(2-ethoxycarbonylethyl)-L-argininamide, N²-dansyl-N-allyl-L-argininamide, N²-dansyl-N-methyl-N-(n-butyl)-L-argininamide, N²-dansyl-N-methyl-N-(2-methoxycarbonylethyl)-L-argininamide, N²-dansyl-N-methyl-N-benzyl-L-argininamide, N²-dansyl-N-methyl-N-(2-acetylethyl)-L-argininamide, 1-(N²-dansyl-L-arginyl)pyrrolidine, 1-(N² -dansyl-L-arginyl)piperidine,2-methyl-1-(N² -dansyl-L-arginyl)piperidine, 3-methyl-1-(N²-dansyl-L-arginyl)piperidine, 4-methyl-1-(N²-dansyl-L-arginyl)piperidine, 4-ethyl-1-(N²-dansyl-L-arginyl)piperidine, 4-(n-propyl)-1-(N²-dansyl-L-arginyl)piperidine, 4-(isopropyl)-1-(N²-dansyl-L-arginyl)piperidine, 4-methoxycarbonyl-1-(N²-dansyl-L-arginyl)piperidine, 4-acetyl-1-(N²-dansyl-L-arginyl)piperidine, N²-dansyl-N,N-hexamethylene-L-argininamide, N²-dansyl-N,N-heptamethylene-L-argininamide, N²-dansyl-N,N-octamethylene-L-argininamide, 4-(N²-dansyl-L-arginyl)morpholine, 2-(N² -dansyl-L-arginyl)isoindoline,4-methyl-1-(N² -dansyl-L-arginyl)piperazine, and N²-dansyl-N-(1,2,3,4-tetrahydro-1-quinolyl)argininamide.

These particular compounds are highly potent in their antithromboticactivity.

For the preparation of the compounds of this invention, various methodscan be employed depending upon the particular starting materials and/orintermediates involved. Successful preparation of these compounds ispossible by way of several synthetic routes which are outlined below.

(1) Preparation of N² -dansyl-L-arginine esters

N² -dansyl-L-arginine, which is the starting material for thepreparation of N² -dansyl-L-arginine esters, is most generally obtainedby reacting L-arginine and dansyl chloride, that is,5-dimethylamino-1-naphthalenesulfonyl chloride, in the presence of abase. However, N² -dansyl-L-arginine may also be obtained by reactingornithine, the ω-position of which is protected, with dansyl chloride inthe presence of a base, removing the protective group at the ω-positionof the product, and thereafter guanidylating the obtained N² -dansylornithine by conventional procedures.

N² -dansyl-L-arginine esters or acid addition salts thereof are preparedby esterifying the above-mentioned N² -dansyl-L-arginine in accordancewith the processes explained below.

(a) Esterification of N² -dansyl-L-arginine with an alcohol

(i) Esterification by heating N² -dansyl-L-arginine and an alcohol

The reaction rate is low in this method which is therefore conductedunder high pressure at an elevated temperature. Care must be exercised,since N² -dansyl-L-arginine is easily decomposed at high temperatures.

(ii) Esterification of N² -dansyl-L-arginine with an alcohol in thepresence of an esterification catalyst

Suitable esterification catalysts include hydrogen halides, such ashydrogen chloride, hydrogen bromide or the like; mineral acids such assulfuric acid, nitric acid, phosphoric acid, or the like; organic acids,such as toluenesulfonic acid, benzenesulfonic acid, methanesulfonicacid, trifluoromethanesulfonic acid, trifluoroacetic acid, cationic ionexchange resins or the like; and Lewis acids, such as boron trifluoride,aluminum chloride, or the like. Strong acids are especially suitable.

A strong acid esterification catalyst adds to an N² -dansyl-L-arginineester to form an acid addition salt thereof. Normally, 2 equivalents ofacid add to 1 equivalent of N² -dansyl-L-arginine ester, and therefore,2 equivalents or more of the esterification catalyst are preferably usedfor each 1 equivalent of N² -dansyl-L-arginine.

Suitable alcohols for the above-mentioned esterification includeprimary, secondary, and tertiary alkyl alcohols containing up to 10carbon atoms, such as methanol, ethanol, n-propanol, isopropyl alcohol,tert-butyl alcohol, n-amyl alcohol, n-hexyl alcohol, 2-ethylhexanol;aralkyl alcohols containing up to 15 carbon atoms, such as benzylalcohol, phenethyl alcohol, 1-phenylethanol, 1-phenyl-1-propanol, or thelike; tetrahydrofurfuryl alcohol; alkenyl alcohols containing up to 10carbon atoms, such as allyl alcohol, crotyl alcohol, methyl vinylcarbinol, or the like; alkynyl alcohols containing up to 10 carbonatoms, such as propargyl alcohols, 3-butyn-1-ol, or the like; cycloalkylalcohols containing up to 10 carbon atoms, such as cyclohexanol,cyclopentanol, or the like; and alkyl alcohols containing up to 10carbon atoms substituted by an alkoxy group of up to 10 carbon atoms, ahalogen or a nitro group, such as 3-chloro-1-propanol,2-chloro-1-propanol, 1-chloro-2-propanol, 2-fluoro-1-ethanol,2-chloro-1-ethanol, 4-chloro-1-butanol, 2-nitro-1-butanol,3-nitro-1-propanol, 2-methoxyethanol, 3-ethoxypropanol, or the like.

N² -dansyl-L-arginine reacts with an equimolar amount of an alcohol.However, at least 5 moles of alcohol per mole of N² -dansyl-L-arginineare preferably employed to enhance the reaction rate.

The esterification reaction can be carried out in an inert reactionsolvent, such as an aromatic hydrocarbon, e.g., benzene, toluene,xylene, or the like; a chlorinated hydrocarbon, e.g., carbontetrachloride, chloroform, dichloromethane, or the like; a hydrocarbonsolvent, e.g., hexane, cyclohexane, or the like; an ether, e.g.,dioxane, tetrahydrofuran, or the like; or a mixture of these compounds.Especially preferred solvents include benzene, toluene, xylene,cyclohexane, carbon tetrachloride, dichloromethane, which formazeotropic mixtures with water, and are therefore advantageous for theesterification reaction, since water formed during the reaction can beeasily removed, and the reaction can be carried out advantageously atequilibrium.

The reaction temperature is dependent upon the alcohol to be employedand the activity of the catalyst. Generally, the temperature ranges from0° C. to the boiling point of the alcohol or solvent. The reaction timevaries widely with the species of alcohol employed and the activity ofthe catalyst and ranges from 10 minutes to 15 hours.

After the reaction is completed, the alcohol and/or solvent is distilledoff, and an N² -dansyl-L-arginine ester or an acid addition salt thereofis obtained. Normally, 2 equivalents of acid esterification catalystaddes to the N² -dansyl-L-arginine ester. The acid addition salt can beconverted to the corresponding N² -dansyl-L-arginine ester by adjustingthe pH of the medium.

N² -dansyl-L-arginine esters and acid addition salts thereof can bepurified by recrystallization from a combination of solvents, such asethyl ether, alcohols, acetone or the like, or reprecipitating byaddition of ether to an alcohol solution of the compounds.

(iii) Esterification of N² -dansyl-L-arginine by the reaction of N²-dansyl-L-arginine with an alcohol and a thionyl halide

Suitable thionyl halides include thionyl chloride and thionyl bromide.N² -dansyl-L-arginine reacts with an equimolar amount of thionyl halide.However, at least 2 moles of thionyl halide per one mole of N²-dansyl-L-arginine are desirable in order to drive the reaction tocompletion. During the reaction, the thionyl halide decomposes to ahydrogen halide and SO₂, and the formed hydrogen halide adds to the N²-dansyl-L-arginine ester to form a dihalogeno acid salt of the N²-dansyl-L-arginine ester.

The other reaction conditions and the procedures for separation andpurification of the product are the same as in process (ii)(esterification with an esterification catalyst).

(iv) Preparation of N² -dansyl-L-arginine methyl ester

N² -dansyl-L-arginine methyl ester can be prepared by the reaction of N²-dansyl-L-arginine with diazomethane; reaction of N² -dansyl-L-argininewith dimethyl sulfite and tosylsulfonic acid; and reaction of N²-dansyl-L-arginine with dimethyl sulfate.

(v) Reaction of an alkali metal salt of N² -dansyl-L-arginine with analkyl halide

Alkyl esters of N² -dansyl-L-arginine can be prepared by reacting analkali metal salt of N² -dansyl-L-arginine and an alkyl halide in apolar solvent.

In addition, N² -dansyl-L-arginine may be esterified by other processes,but processes (ii) and (iii) are generally used.

(vi) Reaction of an L-arginine ester with a dansyl halide

L-arginine esters or acid addition salts thereof, which are used as thestarting materials of N² -dansyl-L-arginine esters or acid additionsalts thereof, are most generally obtained by reacting L-arginine withan alcohol in the presence of an acid catalyst. When the esterificationis carried out in the presence of an acid catalyst, an acid additionsalt of an L-arginine ester is usually obtained.

Suitable dansyl halides includes dansyl chloride, dansyl bromide or thelike. Dansyl chloride is preferred.

The reaction between an L-arginine ester or an acid addition saltthereof and a dansyl halide is normally carried out in the presence of abase. The base captures the hydrogen halide formed during the reactionand enhances the reaction rate.

Suitable bases include organic bases, such as triethylamine, pyridine,or the like; and common inorganic bases such as sodium hydroxide,potassium hydroxide, potassium carbonate, or the like. The inorganicbases are usually used in aqueous solution.

The base is normally used in an amount at least equivalent to theL-arginine ester. When an acid addition salt of an L-arginine ester isused as the starting material, an excess of base sufficient to convertthe L-arginine ester acid addition salt to the L-arginine ester isdesirably used in addition to the amount to be used as the catalyst.

The dansyl halide reacts with an equimolar amount of an L-arginine esteror an acid addition salt thereof. The reaction between an L-arginineester or an acid addition salt thereof and a dansyl halide is usuallycarried out in a solvent. Suitable solvents include water, chlorinatedsolvents, such as dichloromethane, chloroform, carbon tetrachloride, andthe like; aromatic hydrocarbons, such as benzene, toluene, xylene andthe like; esters such as ethyl ether, tetrahydrofuran, tetrahydropyranand the like; ketones such as acetone, methyl ethyl ketone,cyclohexanone and the like; basic solvents, such as dimethylacetamide,dimethylformamide, tetramethylurea, N-methylpyrrolidone, pyridine,quinoline, and the like; or a mixture of two or more of these solvents.A basic solvent acts as an acid acceptor, and therefore addition of baseis unnecessary when such solvent is used.

The reaction temperature is dependent on the species of arginine estersand bases, but is generally between 0° C. and the boiling temperature ofthe solvent employed.

The reaction time varies with the species of the arginine ester and isusually between 10 minutes and 15 hours.

After the reaction is completed, the produced salt is washed away withwater, the solvent is removed by distillation, and the obtained productis washed with water and/or the solvent. To the thus obtained N²-dansyl-L-arginine ester, ether and an acid (e.g., hydrochloric acid,p-toluene-sulfonic acid, or the like) are added, and the formed acidaddition of salt of N² -dansyl-L-arginine ester is isolated.

(2) Preparation of N² -dansyl-L-argininamides (a) Reaction of an N²-dansyl-L-arginine ester with a primary amine

Suitable N² -dansyl-L-arginine esters or the acid addition saltsthereof, include the methyl ester, ethyl ester, isopropyl ester and thelike or the hydrochlorides thereof. Suitable amines include primaryamines, such as an alkylamine containing not more than 10 carbon atoms,e.g., methylamine, ethylamine, n-propylamine, isopropylamine,n-butylamine, n-hexylamine, n-heptylamine and the like; an aralkylaminecontaining not more than 10 carbon atoms, e.g., β-phenylethylamine andthe like; a cycloalkylamine containing not more than 10 carbon atoms,e.g., cyclopropylamine, cyclobutylamine, cyclopentylamine,cyclohexylamine, cyclooctylamine, 4-methylcyclohexylamine, and the like;an alkylamine containing not more than 10 carbon atoms substituted by analkoxy group containing not more than 10 carbon atoms, e.g.,2-methoxyethylamine, 2-ethoxyethylamine, 3-methoxypropylamine,3-methoxybutylamine, 3-methoxypentylamine and the like; acycloalkylalkylamine containing not more than 10 carbon atoms, e.g.,cyclohexylmethylamine, 2-cyclohexylethylamine, cyclobutylmethylamine,3-cyclopentylbutylamine and the like, and an alkenylamine containing notmore than 10 carbon atoms, e.g., allylamine, crotylamine, 3-butenylamineand the like.

The amine is preferably used in an amount equivalent to or greater thanthe N² -dansyl-L-arginine ester. The amine is preferably used in excessin order to enhance the reaction rate and to carry out the reactionadvantageously at eqyilibrium. The amine is usually used in an amount 2to 10 times the molar quantity of the N² -dansyl-L-arginine ester. Whenan acid addition salt of an N² -dansyl-L-arginine ester is used, theamine is usually converted to an acid addition salt. Therefore, it isnecessary to use an amine corresponding to the acid addition salt of theamine to be formed in excess.

A basic compound may be used as a catalyst. Specifically, an alkalimetal alkoxide, such as sodium methoxide or a tertiary amine, such aspyridine or the like are preferable. When these catalysts are used, thereaction rate is enhanced and therefore the amine can be used in alesser amount and milder reaction conditions are thus possible.

If the amine is used in large excess, N² -dansyl-L-arginine esters oracid addition salts thereof will dissolve in the amine, and thereforethe reaction will proceed without a solvent. However, solvents, such asalcohols, e.g., methanol, ethanol, butanol and the like, ethers, e.g.,ethyl ether, tetrahydrofuran, tetrahydropyran, dioxane and the like;hydrocarbons, e.g., benzene, toluene, cyclohexane and the like;halogenated hydrocarbons, e.g., carbon tetrachloride, chloroform,dichloromethane and the like; and water can be used.

The reaction is usually carried out by mixing an N² -dansyl-L-arginineester or an acid addition salt thereof with an excess amount of anamine, the resulting homogeneous solution is allowed to stand at roomtemperature. However, the reaction mixture can be heated to atemperature up to the boiling temperature of the amine or solvent toenhance the reaction rate.

The reaction time is dependent on the basicity and amount of amineemployed, among other factors, but usually ranges from several hours toseveral days.

After the reaction is completed, the product is collected by filtration,washed with water, and purified by recrystallization from a suitablesolvent, e.g., aqueous methanol, or the like. If solid product does notform, the excess amine and/or the solvent is removed by distillation,and the residue is washed and purified by recrystallization from asuitable solvent.

(b) Reaction of an L argininamide with a dansyl halide

An L-argininamide or an acid addition salt thereof can be obtained byprotecting the guanidino and α-amino group of the arginine vianitration, acetylation, formylation, phthaloylation,trifluoroacetylation, p-methoxybenzyloxycarbonylation, benzoylation,benzyoxycarbonylation, t-butyloxycarbonylation or tritylation and thencondensing the obtained arginine derivative with an amine by aconventional process such as the acid chloride process, acid azideprocess, mixed acid anhydride process, activated esterification process,carbodiimide process, or the like, and thereafter removing theprotective group.

Suitable dansyl halides include dansyl chloride, dansyl bromide or thelike, but dansyl chloride is preferred.

The reaction between an L-argininamide or an acid addition salt thereofand a dansyl halide is usually carried out in the presence of a base.The basic compound captures the hydrogen chloride, which is formedduring the reaction, and thus promotes the reaction.

Suitable bases include organic bases such as triethylamine, pyridine andthe like; or inorganic bases, such as sodium hydroxide, potassiumhydroxide, potassium carbonate and the like. Inorganic bases are usuallyused in aqueous solution.

The base is used in excess of the amount equivalent to theL-argininamide. When an acid addition salt of an L-argininamide is used,a base is preferably used in an amount sufficient to convert the acidaddition salt of the L-argininamide to the free L-argininamide inaddition to the amount of base to be used as the catalyst.

A dansyl halide is usually reacted with an equimolar amount of anL-argininamide or an acid addition salt thereof in a solvent. Suitablesolvents include chlorinated hydrocarbons such as dichloromethane,chloroform, carbon tetrachloride and the like; aromatic hydrocarbons,such as benzene, toluene, xylene and the like; ethers, such as ethylether, tetrahydrofuran, dioxane, tetrahydropyran and the like; ketones,such as acetone, methyl ethyl ketone, cyclohexanone and the like; basicsolvents, such as dimethyl acetamide, dimethylformamide,tetramethylurea, N-methylpyrrolidone, pyridine, quinoline and the like;or a mixture of two or more of these solvents. A basic solvent acts asan acid acceptor, and therefore addition of further base is not requiredin these instances.

The reaction temperature is dependent on the species of theL-argininamide and base, but usually ranges between 0° C. and theboiling temperature of the solvent. The reaction time varies with thespecies of the L-argininamide and is usually between 10 minutes and 15hours.

After the reaction is completed, the formed salt is removed by washingwith water, the solvent is removed by distillation, and the obtainedproduct is washed with water and/or the solvent, and the N²-dansyl-L-argininamide is obtained. The thus obtained N²-dansyl-L-argininamide can be isolated in the form of an acid additionsalt thereof by addition of ethyl ether and an acid (e.g., hydrochloricacid, p-toluenesulfonic acid, and the like).

(c) Elimination of the N^(G) -substituent from an N^(G) -substituted-N²-dansyl-L-argininamide having the formula (II) ##STR22##

An N² -dansyl-L-argininamide is prepared by eliminating the N^(G)-substituent from an N^(G) -substituted-N² -dansyl-L-argininamide havingthe above formula (II) by decomposition with an acid or by means ofhydrogenation. In the formula (II), R is the same as in the formula (I),X and Y are hydrogen and protective groups for the guanidino group. Atleast one of X and Y is nitro, tosyl, trityl and oxycarbonyl. Specificexamples of R in the formula (II) are as follows:

(1) In the case where R= ##STR23## R₁ and R₂ are respectively an alkylgroup containing not more than 10 carbon atoms, e.g., methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, n-hexyl, n-heptyl andthe like; an aryl group containing not more than 10 carbon atoms, e.g.,phenyl, tolyl and the like; an aralkyl group containing not more than 10carbon atoms, e.g., benzyl, phenethyl, 3-phenylpropyl and the like; acycloalkyl group containing not more than 10 carbon atoms, e.g.,cyclopropyl, cyclohexyl and the like; a cycloalkylalkyl group containingnot more than 10 carbon atoms, e.g., cyclohexylmethyl,3-cyclohexylpropyl and the like; an alkenyl group containing not morethan 10 carbon atoms, e.g., allyl, crotyl, 2-hexenyl and the like; analkyl group containing not more than 10 carbon atoms substituted by analkoxy group, an alkoxycarbonyl group, an acyl group, an acyloxy group,an arylcarbamoyl group or an N,N-polymethylenecarbamoyl group,respectively containing not more than 10 carbon atoms or a carboxylgroup, e.g., methoxyethyl, methoxypropyl, ethoxyethyl,ethoxycarbonylmethyl, 2-ethoxycarbonylethyl, 2-methoxycarbonylethyl,3-ethoxycarbonylpropyl, 2-acetylethyl, 2-acetoxyethyl,2-phenylcarbamoylethyl, or N,N-tetramethylenecarbamoylmethyl.

(2) In the case where R= ##STR24## R may be a 1-polymethyleneiminylgroup or an oxo-substituted group thereof containing not more than 10carbon atoms, a 1-polymethyleneiminyl group containing not more than 10carbon atoms substituted with an alkyl, acyl, alkoxy, or alkoxycarbonylgroup containing not more than 10 carbon atoms, e.g., azetidinyl,3-methoxy-1azetidinyl, 3-ethoxy-1-azetidinyl, 1-pyrrolidinyl,2-ethoxycarbonyl-1-pyrrolidinyl, 1-(2-pyrrolidonyl), 1-piperidino,1-(4-piperidonyl), 4-methyl-1-piperidino, 4-ethyl-1-piperidino,4-n-propyl-1-piperidino, 4-isopropyl-1-piperidino,2-methyl-1-piperidino, 3-methyl-1-piperidino,2-ethoxycarbonyl-1-pyrrolidinyl, 4-methoxy-1-piperidino,4-oxo-1-piperidino, 4-acetyl-1-piperidino,4-methoxycarbonyl-1-piperidino, 4-carbamoyl-1-piperidino,1-hexamethyleneiminyl, 1-octamethyleneiminyl and the like; an oxazole orthiazole, such as 3-oxazolidinyl, 3-thiazolidinyl, and the like; anisoxazole or isothiazole, such as 2-isoxazolidinyl, 2-isothiazolidinyl,and the like; an oxazine, such as 4-morpholino,2,6-dimethyl-4-morpholino, and an oxazine group represented byn-(tetrahydro-1,n-oxazinyl), such as 3-(tetrahydro-1,3-oxazinyl and thelike; a thiazine, such as 4-(tetrahydro-1,4-thiazinyl) and the like;4-methyl-1-piperazinyl, 4-acetyl-1-piperazinyl, 1-piperazinyl,2-isoindolinyl, 1-indolinyl, 1,2,3,4-tetrahydro-2-isoquinolyl,4(4-azabicyclo-[3,2,2]-nonyl), 1,2,3,4-tetrahydro-1-quinolyl and thelike.

In formula (II), X and Y respectively represent a hydrogen atom or aprotective group for the guanidino group, and at least one of X and Y isa protective group for the guanidino group. Suitable protective groupsinclude nitro, tosyl, trityl, or an oxycarbonyl, such asbenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,tert-butyloxycarbonyl and the like.

The N^(G) -substituted-N² -dansyl-L-argininamides as represented bygeneral formula (II) or acid addition salts thereof can be obtained bycondensing an N^(G) -substituted-N² -substituted-arginine (usually theN² -substituent is a protective group for the amino group, such asbenzyloxycarbonyl, t-butyloxycarbonyl, or the like) and an amine via theacid azide process, mixed acid anhydride process, activatedesterification process, carbodiimido process or the like, selectivelyremoving only the N² -substituent by means of catalytic hydrogenation oracid decomposition, and reacting the thus obtained N^(G)-substituted-L-argininamide or an acid addition salt thereof with adansyl halide, such as dansyl chloride in the presence of a base in asolvent. Suitable bases include organic bases, such as triethylamine,pyridine and the like; or inorganic bases, such as sodium hydroxide,potassium hydroxide, potassium carbonate, sodium hydrogen carbonate andthe like. Inorganic bases are normally used in aqueous solution.

The base is preferably used in an amount not less than the amountequivalent to the N^(G) -substituted-L-argininamide. When an acidaddition salt of an N^(G) -substituted-L-argininamide is used, the baseis preferably used in an amount sufficient to neutralize the acidaddition salt in addition to the amount to be used as the catalyst. Thedansyl halide is normally used in an equimolar amount.

Suitable solvents include water; chlorinated solvents, such asdichloromethane, chloroform, carbon tetrachloride and the like; aromatichydrocarbons, such as benzene, toluene, xylene and the like; ethers,such as ethyl ether, tetrahydrofuran, dioxane and the like; ketones,such as acetone, methyl ethyl ketone, cyclohexanone, and the like; basicsolvents, such as dimethylacetamide, dimethylformamide, tetramethylurea,N-methylpyrrolidone, pyridine, quinoline, and the like; or a mixture oftwo or more of the above-mentioned solvents. A basic solvent acts as anacid acceptor, and therefore further addition of base is not required inthese instances.

The reaction temperature is dependent on the species of N^(G)-substituted-L-argininamide and base, but usually ranges from -10° C. tothe boiling temperature of the solvent.

The reaction time varies with the species of N^(G)-substituted-L-argininamide and base, and the reaction temperature, andis usually from 5 minutes to 24 hours.

After the reaction is completed, the solvent and base are distilled off,the formed salt is removed by washing with water, and the N^(G)-substituted-N² -dansyl-L-argininamide is purified by recrystallizing orreprecipitating from a suitable solvent. The reaction product may alsobe separated and purified by means of chromatography. Suitable elutantsinclude chlorinated solvents, such as chloroform, dichloromethane andthe like; a chlorinated solvent containing alcohol and the like.

As explained above, N² -dansyl-L-argininamide represented by generalformula (II) or an acid addition salt thereof is obtained from N^(G)-substituted-N-² -dansyl-L-argininamide by removing the N^(G)-substituent, which is a protective group for the guanidino group of theamide, via hydrogenation or acid decomposition.

Suitable acids for the acid decomposition include hydrogen halides, suchas hydrogen chloride, hydrogen bromide, hydrogen fluoride; and organicacids, such as trifluoroacetic acid, trifluoromethanesulfonic acid,formic acid, acetic acid, and the like.

The acid decomposition is preferably carried out by treating an N^(G)-substituted-N² -dansyl-L-argininamide or an acid addition salt thereofwith any of the above-mentioned acids without a solvent or in a solvent,such as an alcohol, e.g., methanol, ethanol and the like; an ether,e.g., tetrahydrofuran, dioxane, and the like; an organic acid, e.g.,acetic acid and the like; or an ester, e.g., ethyl acetate and the like,at a temperature between 31 10° C. and 100° C., preferably at roomtemperature. The time required for the acid decomposition varies withthe species of the acid and solvent, the protective N^(G) -substituent,and the temperature of treatment, and is from 30 minutes to 24 hours.

After the decomposition is completed, the N² -dansyl-L-argininamide oran acid addition salt thereof is obtained by removing the solvent andthe excess acid or adding to the reaction mixture an inert solvent, suchas ethyl ether, petroleum ether, a hydrocarbon solvent, or the like soas to form a precipitate and collecting the precipitate. An excess ofacid is usually used, and therefore the N² -dansyl-L-argininamide whichis obtained by removing the protective group is in the form of an acidaddition salt. This salt can be easily converted to a free amide byneutralization.

Hydrogenation can be carried out according to the general procedures ofreductive hydrogenation, although catalytic hydrogenation is mostadvantageous. Catalytic hydrogenation is carried out in the presence ofa hydrogen-activating catalyst in a hydrogen atmosphere. Suitablehydrogen-activating catalysts include Raney nickel, palladium, platinumand the like. Suitable solvents include alcohols, such as methanol,ethanol and the like; ethers, such as dioxane, tetrahydrofuran and thelike; organic acids, such as acetic acid, propionic acid and the like;or a mixture of two or more of the above-mentioned solvents.

The reaction temperature is dependent on the protective group for theguanidino group and the activity of the employed catalyst, and isusually between 0° C. and the boiling temperature of the solvent. Thehydrogen pressure is dependent on the reaction temperature and activityof the employed catalyst. Atmospheric pressure is sufficient for thereaction. The reaction time is dependent on the activity of thecatalyst, the reaction temperature, hydrogen pressure and the like andis usually from 2 hours to 120 hours.

After the hydrogenation is finished, the catalyst is removed byfiltration, the solvent is removed by distillation, and the N²-dansyl-L-argininamide or an acid addition salt thereof is obtained. Theacid addition salt is easily converted to the free N²-dansyl-L-argininamide by neutralization.

The thus obtained N² -dansyl-L-argininamide or acid addition saltthereof is purified by recrystallizing from a solvent which is a mixtureof two or more of the following: water, ethyl ether, alcohols, acetone,or the like, or by reprecipitating by addition of ethyl ether to analcohol solution of the compound.

N² -dansyl-L-arginine esters and amides of this invention having theformula (I) form acid addition salts with any of a variety of inorganicand organic acids. The product of the reactions described above can beisolated as the free base or as the acid addition salt. In addition, theproduct can be obtained as pharmaceutically acceptable acid additionsalts by reacting one of the free bases with an acid, such ashydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric,acetic, citric, maleic, succinic, lactic, tartaric, gluconic, benzoic,methanesulfonic, ethanesulfonic, benzensulfonic, p-toluenesulfonic acidor the like.

As stated above, N² -dansyl-L-arginine esters and amides, and acidaddition salts thereof of this invention are characterized by highlyspecific inhibitory activity against thrombin, and therefore thesecompounds are useful in the determination of thrombin in blood asdiagnostic reagents, and/or for the medical control or prevention ofthrombosis.

The antithrombotic activities of the N² -dansyl-L-arginine derivativesof this invention were compared with those of a known antithromboticagent, (N² -(p-tolylsulfonyl)-L-arginine methyl ester), by determiningthe fibrinogen coagulation time. The measurement of the fibrinogencoagulation time was conducted as follows:

An 0.8 ml aliquot of a fibrinogen solution, which had been prepared bydissolving 150 mg of bovine fibrinogen (Cohn fraction I) supplied byArmour Inc. in 40 ml of a borate saline buffer (pH 7.4), was mixed with0.1 ml of a borate saline buffer, pH 7.4, (control) or a sample solutionin the same buffer, and 0.1 ml of a thrombin solution (5 units/ml)supplied by Mochida Pharmaceutical Ltd. was added to the solutions in anice bath. Immediately after mixing, the reaction mixture was transferredfrom the ice bath to a bath thermostated at 25° C. Coagulation timeswere taken as the period between the time of transference to the 25° C.bath and the time of the first appearance of fibrin threads. In thecases where no drug samples were added, the coagulation time was 50-55seconds.

The experimental results are summarized in Table 1. The term"concentration required to prolong the coagulation time by a factor oftwo" is the concentration of an active ingredient require to prolong thenormal coagulation time 50-55 seconds to 100-110 seconds.

The inhibitors are shown in Table 1 by indicating R in the generalformula (II) and the added acid and/or water of crystallization.

When a solution containing an N² -dansyl-L-arginine derivative of thisinvention was administered intravenously into animal bodies, the highantithrombotic activity in the circulating blood was maintained for fromone to three hours. The halflife for decay of the antithromboticcompounds of this invention in circulating blood was shown to beapproximately 30 minutes; the physiological conditions of the hostanimals were well maintained. The experimental decrease of fibrinogen inanimals caused by infusion of thrombin was satisfactorily controlled bysimultaneous infusion of the compounds of this invention.

The acute toxicity values (LD₅₀) determined by oral administration ofsubstances of formula (I) in mice range from about 1,000 to 10,000milligrams per kilogram of body weight. Representative LD₅₀ values, forexample, for N² -dansyl-L-arginine n-butyl ester, 4-methyl-1-(N²-dansyl-L-arginyl)piperidine, 4-ethyl-1-(N²-dansyl-L-arginyl)piperidine, 2-(N² -dansyl-L-arginyl)-isoindolineare >6000, 1310, 1375, 1360 milligrams per kilogram, respectively.

The therapeutic agents of this invention may be administered alone or incombination with pharmaceutically acceptable carriers, the proportion ofwhich is determined by the solubility and chemical nature of thecompound, chosen route of administration and standard pharmaceuticalpractice. For example, the compounds may be injected parenterally, thatis, intramuscularly, intravenously or subcutaneously. For parenteraladministration, the compounds may be used in the form of sterilesolutions containing other solutes, for example, sufficient saline orglucose to make the solution isotonic. The compounds may be administeredorally in the form of tablets, capsules, or granules containing suitableexcipients such as starch, lactose, white sugar and the like. Thecompounds may be administered sublingually in the form of troches orlozenges in which each active ingredient is mixed with sugar or cornsyrups, flavoring agents and dyes, and then dehydrated sufficiently tomake the mixture suitable for pressing into solid form. The compoundsmay be administered orally in the form of solutions which may containcoloring and flavoring agents.

Physicians will determine the dosage of the present therapeutic agentswhich will be most suitable, and dosages vary with the mode ofadministration and the particular compound chose. In addition, thedosage will vary with the particular patient under treatment.

When the composition is administered orally, a larger quantity of theactive agent will be required to produce the same effect as caused witha smaller quantity given parenterally. The therapeutic dosage isgenerally 10-50 mg/kg of active ingredient parenterally, 10-500 mg/kgorally per day.

Having generally described the invention, a more complete understandingcan be obtained by reference to certain specific examples, which areincluded for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLE 1

To an ice-cooled suspension of 1.0 gram of N² -dansyl-L-arginine in 15ml of methanol was added dropwise 0.5 ml of thionyl chloride withvigorous stirring. After being allowed to stand for 2 hours at roomtemperature, the reaction mixture was refluxed for 2 hours, and wasevaporated to dryness (syrup). Treatment of the residual syrup with coldethyl ether and a small amount of water gave crude crystals. Afterrecrystallization from methanol-ethyl ether, colorless N²-dansyl-L-arginine methyl ester dihydrochloride monohydrate was obtainedin 92% yield: mp. 147°-150° C.

    ______________________________________                                        Elemental analysis (as C.sub.19 H.sub.29 O.sub.4 N.sub.5 S.HCl.H.sub.2        O)                                                                                     C           H      N                                                 ______________________________________                                        Calculated:                                                                              44.62         6.11   13.69                                         Found:     44.42         6.17   13.64                                         ______________________________________                                    

EXAMPLE 2

A suspension of 1.0 gram of N² -dansyl-L-arginine in 15 ml of anhydrousethanol was saturated with dry hydrogen chloride for 1 hour. Thereaction mixture was reflexed for an additional one hour. After cooling,the reaction mixture was concentrated in vacuo. The residue wastriturated with cold ethyl ether to give a crystalline product. Aftercrystallization from ethanol-ethyl ether, N² -dansyl-L-arginine ethylester dihydrochloride was obtained in 95% yield; mp. 140°-144° C.

    ______________________________________                                        Elemental analysis (as C.sub.20 H.sub.29 O.sub.4 N.sub.5 S.2HCl)                       C           H      N                                                 ______________________________________                                        Calculated:                                                                              47.24         6.15   13.77                                         Found:     46.95         6.21   14.00                                         ______________________________________                                    

EXAMPLE 3

A mixture of 1.0 grams of N² -dansyl-L-arginine and 1.4 gram ofp-toluenesulfonic acid monohydrate in 10 ml of benzyl alcohol was heatedfor 30 minutes at 100° C. To the thus obtained clear solution, 100 ml ofbenzene was added, and the mixture was refluxed for 5 hours, removingwater by azeotropic distillation. After the solvent was removed bydistillation, 100 ml of ethyl ether was added to the residue, giving acrystalline mass. Crystallization from acetone gave N²-dansyl-L-arginine benzyl ester di(p-toluenesulfonate) in 87% yield; mp.150°-153° C.

    ______________________________________                                        Elemental analysis (as C.sub.25 H.sub.31 O.sub.4 N.sub.5 S.C.sub.14           H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              55.63         5.63   8.32                                          Found:     55.33         5.61   8.19                                          ______________________________________                                    

EXAMPLE 4

A mixture of 1.0 gram of N² -dansyl-L-arginine and 1.4 gram ofp-toluenesulfonic acid monohydrate in 10 ml of 2-ethylhexanol was heatedfor 30 minutes at 100° C. To the thus obtained clear solution, 100 ml ofbenzene was added, and the mixture was refluxed for 10 hours, removingwater by azeotropic distillation. After the solvent was removed bydistillation, 100 ml of ethyl ester was added to the residue to give acrystalline mass. Crystallization from acetone gave N²-dansyl-L-arginine 2-ethylhexyl ester di(p-toluenesulfonate) in 91%yield; mp. 170°-174° C.

    ______________________________________                                        Elemental analysis (as C.sub.26 H.sub.41 O.sub.4 N.sub.5 S.C.sub.14           H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              55.60         6.65   8.11                                          Found:     55.37         6.59   8.18                                          ______________________________________                                    

EXAMPLE 5

To an ice-cooled suspension of 1.0 gram of N² -dansyl-L-arginine in 15ml of isopropanol was added dropwise 0.5 ml of thionyl chloride withvigorous stirring. After being allowed to stand for 2 hours at roomtemperature, the reaction mixture was refluxed for 4 hours, and wasevaporated to dryness (syrup). Treatment of the residual syrup with coldethyl ether gave crude crystals. After recrystallization fromisopropanol-ethyl ether, colorless N² -dansyl-L-arginine isopropyl esterdihydrochloride was obtained in 90% yield; mp. 110°-120° C.

    ______________________________________                                        Elemental analysis (as C.sub.21 H.sub.31 O.sub.4 N.sub.5 S.2HCl)                       C           H      N                                                 ______________________________________                                        Calculated:                                                                              48.27         6.37   13.40                                         Found:     47.90         6.08   13.21                                         ______________________________________                                    

EXAMPLE 6

A mixture of 1.0 gram of N² -dansyl-L-arginine and 1.4 gram ofp-toluenesulfonic acid monohydrate in 10 ml of n-hexyl alcohol washeated for 30 minutes at 100° C. To the thus obtained clear solution,100 ml of benzene was added, and the mixture was refluxed for 3 hours,removing water by azeotropic distillation. After the solvent was removedby distillation, 50 ml of ethyl ether and 50 ml of petroleum ether wereadded to the residue to give a crystalline mass. Crystallization fromacetone gave N² -dansyl-L-arginine-n-hexyl ester di(p-toluenesulfonate)in 95% yield; mp. 190°-193° C.

    ______________________________________                                        Elemental analysis (as C.sub.24 H.sub.30 O.sub.4 N.sub.5 S.C.sub.14           H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.59         6.39   8.38                                          Found:     54.33         6.48   8.11                                          ______________________________________                                    

EXAMPLE 7

A mixture of 1.0 gram of N² -dansyl-L-arginine and 1.0 gram ofp-toluenesulfonic acid monohydrate in 10 ml of n-butyl alcohol washeated for 30 minutes at 100° C. To the thus obtained clear solution,100 ml of benzene was added, and the mixture was refluxed for 3 hours,removing water by azeotropic distillation. After the solvent was removedby distillation, 50 ml of ethyl ether and 50 ml of petroleum ether wereadded to the residue to give a crystalline mass. Crystallization fromacetone gave N² -dansyl-L-arginine-n-butyl ester di(p-toluenesulfonate)in 95% yield; mp. 160°-164° C.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.33 O.sub.4 N.sub.5 S.C.sub.14           H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              53.51         6.11   8.67                                          Found:     53.32         6.14   8.93                                          ______________________________________                                    

EXAMPLE 8

A mixture of 1.0 gram of N² -dansyl-L-arginine and 1.0 gram ofp-toluenesulfonic acid monohydrate in 10 ml of n-amyl alcohol was heatedfor 30 minutes at 100° C. To the thus obtained clear solution, 100 ml ofbenzene was added, and the mixture was refluxed for 4 hours, removingwater by azeotropic distillation. After the solvent was removed bydistillation, 100 ml of petroleum ether was added to the residue to givea crystalline mass. Crystallization from acetone gave N²-dansyl-L-arginine n-amyl ester di(p-toluenesulfonate) in 96% yield; mp.164°-169° C.

    ______________________________________                                        Elemental analysis (as C.sub.23 H.sub.35 O.sub.4 N.sub.5 S.C.sub.14           H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.06         6.25   8.52                                          Found:     53.86         6.10   8.53                                          ______________________________________                                    

EXAMPLE 9

By the same procedures as described in Example 3, N² -dansyl-L-arginineisobutyl ester di(p-toluenesulfonate) was obtained from N²-dansyl-L-arginine and isobutyl alcohol in 92% yield; mp 146°-151° C.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.33 O.sub.4 N.sub.5 S.C.sub.14           H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              53.51         6.11   8.67                                          Found:     53.54         6.11   8.65                                          ______________________________________                                    

EXAMPLE 10

By the same procedures as described in Example 3, N² -dansyl-L-arginineisopentyl ester di(p-toluenesulfonate) was obtained from N²-dansyl-L-arginine and isopentyl alcohol in 94% yield; mp. 163°-168° C.

    ______________________________________                                        Elemental analysis (as C.sub.23 H.sub.35 O.sub.4 N.sub.5 S.C.sub.14           H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.06         6.25   8.52                                          Found:     53.74         6.09   8.57                                          ______________________________________                                    

EXAMPLE 11

A mixture of 1.0 gram of N² -dansyl-L-arginine and 1.4 gram ofp-toluenesulfonic acid monohydrate in 10 ml of 3-chloro-1-propanol washeated for 30 minutes at 100° C. To the thus obtained clear solution,100 ml of benzene was added, and the mixture was refluxed for 5 hours,removing water by azeotropic distillation. After the solvent was removedby distillation, 100 ml of ethyl ether was added to the residue to givea crystalline mass. Crystallization from acetone gave N²-dansyl-L-arginine 3-chloropropyl ester di(p-toluenesulfonate) in 88%yield; mp. 140°-145° C.

    ______________________________________                                        Elemental analysis (as C.sub.21 H.sub.30 O.sub.4 N.sub.5 ClS.C.sub.14         H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              50.74         5.60   8.45                                          Found:     50.30         5.50   8.01                                          ______________________________________                                    

EXAMPLE 12

To a solution of 1.0 gram of L-arginine methyl ester dihydrochloride in50 ml of dichloromethane and 1.15 gram of triethylamine, was added 1.03gram of dansyl chloride with stirring at room temperature. After beingstirred for 5 hours at room temperature, the reaction mixture was pouredinto 30 ml of water.

After separation of the aqueous layer, the dichloromethane solution wasdried over anhydrous Na₂ SO₄. The Na₂ SO₄ was filtered off, and thesolution was evaporated under reduced pressure to give N²-dansyl-L-arginine methyl ester. To the solid was added ethyl estersaturated with dry hydrogen chloride, and N² -dansyl-L-arginine methylester dihydrochloride monohydrate was obtained in 83% yield; mp.147°-150° C. (decomp).

    ______________________________________                                        Elemental analysis (as C.sub.19 H.sub.29 O.sub.4 N.sub.5 S.2HCl.H.sub.2       O)                                                                                     C           H      N                                                 ______________________________________                                        Calculated:                                                                              44.62         6.11   13.69                                         Found:     44.32         6.00   13.39                                         ______________________________________                                    

EXAMPLE 13

To a mixture of 1.5 gram of L-arginine n-butyl ester dihydrochloride and1.4 gram of K₂ CO₃ in 10 ml of water was added dropwise a solution of1.34 gram of dansyl chloride in 20 ml of ethyl ester with vigorousstirring at 0°-5° C. over a period of 30 minutes. After the solution waskept at room temperature for 10 hours with stirring a viscous depositseparated, which was collected and triturated with water and ethylether.

To a suspension of the resulting product in 20 ml of ethyl ether wasadded 2 grams of p-toluenesulfonic acid monohydrate with stirring toyield crystals. Recrystallization from acetone gave 3.60 gram of N²-dansyl-L-arginine n-butyl ester di(p-toluenesulfonate) in 89.1% yield;mp. 160°-163° C.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.33 O.sub.4 N.sub.5 S.C.sub.14           H.sub.16 O.sub.6 S.sub.2)                                                              C           H      N                                                 ______________________________________                                        Calculated:                                                                              53.51         6.11   8.67                                          Found:     53.50         6.20   8.85                                          ______________________________________                                    

EXAMPLE 14

A 1.0 gram amount of N² -dansyl-L-arginine methyl ester dihydrochloridemonohydrate was dissolved in 2 ml of n-butylamine with vigorousagitation. After the resulting solution was allowed to stand at roomtemperature for 2 days, the n-butyl-amine was removed by vacuumdistillation and to the residual syrup was added 5 ml of water to obtaina crystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-(n-butyl-(-L-argininamide monohydrate in90% yield; mp. 150°-152° C.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.34 O.sub.3 N.sub.6 S.H.sub.2 O)                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.98         7.55   17.49                                         Found:     54.72         7.61   17.25                                         ______________________________________                                    

EXAMPLE 15

A 1.0 gram amount of N² -dansyl-L-arginine ethyl ester dihydrochloridewas dissolved in 2 ml of n-propylamine with vigorous agitation. Afterthe resulting solution was allowed to stand at room temperature for 2days, the n-propylamine was removed by distillation in vacuo and to theresidual syrup was added 5 ml of water to obtain a crystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-(n-propyl)-L-argininamide monohydrate in85% yield; mp. 150°-153° C.

    ______________________________________                                        Elemental analysis (as C.sub.21 H.sub.32 O.sub.3 N.sub.6 S.H.sub.2 O)                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.06         7.35   18.02                                         Found:     53.82         7.45   18.13                                         ______________________________________                                    

EXAMPLE 16

A 1.0 gram amount of N² -dansyl-L-arginine methyl ester dihydrochloridemonohydrate was dissolved in 2 ml of isopropylamine with vigorousagitation. After the resulting solution was allowed to stand at roomtemperature for 2 days, the isopropylamine was removed by vacuumdistillation and to the residual syrup was added 5 ml of water to obtaina crystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-isopropyl-L-argininamide in 78% yield; mp.218°-221° C.

    ______________________________________                                        Elemental analysis (as C.sub.21 H.sub.32 O.sub.3 N.sub.6 S)                            C           H      N                                                 ______________________________________                                        Calculated:                                                                              56.23         7.19   18.72                                         Found:     56.38         7.20   18.94                                         ______________________________________                                    

EXAMPLE 17

A 1.0 gram amount of N² -dansyl-L-arginine isopropyl esterdihydrochloride was dissolved in 3 ml of β-phenylethylamine withvigorous agitation. After the resulting solution was allowed to stand atroom temperature for 4 days, the reaction mixture was poured into amixture of 30 ml of water and 30 ml of ethyl ether to obtain acrystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-(β-phenylethyl)-L-argininamide dihydratein 91% yield; mp. 143°-145° C.

    ______________________________________                                        Elemental analysis (as C.sub.26 H.sub.33 O.sub.3 N.sub.6 S.2H.sub.2 O)                 C           H      N                                                 ______________________________________                                        Calculated:                                                                              57.13         7.01   15.38                                         Found:     57.34         6.72   15.64                                         ______________________________________                                    

EXAMPLE 18

To a suspension of 1.0 gram of N² -dansyl-L-arginine methyl esterdihydrochloride monohydrate in 1 ml of tetrahydrofuran was added 3 ml ofethylamine with cooling. The mixture was allowed to stand in a sealedtube at room temperature for three days. After the excess amine wasremoved by evaporation, the residue was poured into 50 ml of water togive a precipitate. Recrystallization from 50% aqueous methanol affordedN² -dansyl-N-ethyl-L-argininamide monohydrate in 93% yield; mp.220°-222° C.

    ______________________________________                                        Elemental analysis: (as C.sub.20 H.sub.30 O.sub.3 N.sub.6 S.H.sub.2 O)                 C           H      N                                                 ______________________________________                                        Calculated:                                                                              53.08         7.13   18.57                                         Found:     53.34         7.18   18.86                                         ______________________________________                                    

EXAMPLE 19

A 1.0 gram amount of N² -dansyl-L-arginine methyl ester dihydrochloridemonohydrate was dissolved in 3 ml of n-hexylamine with vigorousagitation. After the resulting solution was allowed to stand at roomtemperature for 2 days, the n-hexylamine was removed by vacuumdistillation and to the residual syrup was added 20 ml of water toobtain a crystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-(n-hexyl)-L-argininamide monohydrate in93% yield; mp. 133°-135° C.

    ______________________________________                                        Elemental analysis (as C.sub.24 H.sub.38 O.sub.3 N.sub.6 S.H.sub.2 O)                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              56.67         7.93   16.53                                         Found:     56.38         7.59   16.34                                         ______________________________________                                    

EXAMPLE 20

A 1.0 gram amount of N² -dansyl-L-arginine methyl ester dihydrochloridemonohydrate was dissolved in 3 ml of n-heptylamine with vigorousagitation. After the resulting solution was allowed to stand at 80° C.for 5 hours, the reaction mixture was cooled and poured into 30 ml ofcold water to obtain a crystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-(n-heptyl)-L-argininamide in 85% yield;mp. 240°-243° C.

    ______________________________________                                        Elemental analysis (as C.sub.25 H.sub.40 O.sub.3 N.sub.6 S)                            C           H      N                                                 ______________________________________                                        Calculated:                                                                              59.50         7.99   16.66                                         Found:     59.50         7.90   16.68                                         ______________________________________                                    

EXAMPLE 21

A 1.0 gram amount of N² -dansyl-L-arginine methyl ester dihydrochloridemonohydrate was dissolved in 3 ml of isobutylamine with vigorousagitation. After the resulting solution was allowed to stand at roomtemperature for 2 days, the reaction mixture was poured into 20 ml ofwater and was agitated to obtain a crystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-isobutyl-L-argininamide in 80% yield; mp.157°-160° C.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.34 O.sub.3 N.sub.6 S)                            C           H      N                                                 ______________________________________                                        Calculated:                                                                              57.12         7.41   18.17                                         Found:     56.82         7.41   17.90                                         ______________________________________                                    

EXAMPLE 22

A 1.0 gram amount of N² -dansyl-L-arginine methyl ester dihydrochloridemonohydrate was dissolved in 2 ml of 2-methoxyethylamine with vigorousagitation. After the resulting solution was allowed to stand at roomtemperature for 2 days, the 2-methoxy-ethylamine was removed by vacuumdistillation and to the residual syrup was added 5 ml of water to obtaina crystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-(2-methoxyethyl)-L-argininamide dihydratein 90% yield; mp. 130°-135° C.

    ______________________________________                                        Elemental analysis (as C.sub.21 H.sub.32 O.sub.4 N.sub.6 S.2H.sub.2 O)                 C           H      N                                                 ______________________________________                                        Calculated:                                                                              50.38         7.25   16.79                                         Found:     50.03         6.95   16.99                                         ______________________________________                                    

EXAMPLE 23

A 1.0 gram amount of N² -dansyl-L-arginine ethyl ester dihydrochloridewas dissolved in 3 ml of cyclohexylmethylamine with vigorous agitation.After the resulting solution was allowed to stand at 80° C. for 5 hours,the reaction mixture was cooled and poured into 30 ml of water to obtaina crystalline deposit.

The precipitate was collected and recrystallized from 50% aqueousmethanol to give N² -dansyl-N-cyclohexylmethyl-L-argininamide in 85%yield; mp. 253°-256° C.

    ______________________________________                                        Elemental analysis (as C.sub.25 H.sub.38 O.sub.3 N.sub.6 S)                            C           H      N                                                 ______________________________________                                        Calculated:                                                                              59.73         7.62   16.72                                         Found:     59.96         7.48   16.73                                         ______________________________________                                    

EXAMPLE 24

To a solution of 1.0 gram of N-(2-methoxyethyl)-L-argininamide in 30 mlof dichloromethane and 0.52 gram of triethylamine was added 1.16 gram ofdansyl chloride with stirring at room temperature. After stirring for 2hours at room temperature, the dichloromethane was removed bydistillation and the residual syrup was poured into 50 ml of ice water.

After separation of the aqueous layer, the dichloromethane solution wasdried over anhydrous Na₂ SO₄. The Na₂ SO₄ was filtered off, the solutionwas evaporated under reduced pressure, and N²-dansyl-N-(2-methoxyethyl)-L-argininamide dihydrate was obtained in 89%yield; mp. 130°-135° C. (decomp).

    ______________________________________                                        Elemental analysis (as C.sub.21 H.sub.32 O.sub.4 N.sub.6 S.2H.sub.2 O)                 C           H      N                                                 ______________________________________                                        Calculated:                                                                              50.38         7.25   16.79                                         Found:     50.68         6.95   16.99                                         ______________________________________                                    

EXAMPLE 25

To a mixture of 1.0 gram of 1-(L-arginyl)-piperidine and 0.57 gram of K₂CO₃ in 10 ml of water was added dropwise a solution of 1.12 gram ofdansyl chloride in 30 ml of dioxane with vigorous stirring over a periodof 30 minutes while maintaining the temperature at 0° C. The reactionmixture was stirred for an additional 3 hours and the formed precipitatewas removed by filtration. The solvent was evaporated, and to theresidue was added 30 ml of CHCl₃. A small amount of the undissolvedmaterial was filtered and the solution was dried over anhydrous Na₂ SO₄.To the stirred solution was added 20 ml of ether containing 0.5 gram ofacetic acid to precipitate 1-(N² -dansyl-L-arginyl)piperidine diacetate,which was purified by reprecipitation from a methanol-ethyl ethermixture in 72% yield.

    ______________________________________                                        Elemental analysis (as C.sub.23 H.sub.34 O.sub.3 N.sub.6 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.53         7.12   14.13                                         Found:     54.23         7.11   14.43                                         ______________________________________                                    

EXAMPLE 26

A 3.2 gram amount of N^(G) -nitro-N² -(tert-butyloxycarbonyl)-L-argininewas dissolved in a mixture of 40 ml of dry tetrahydrofuran and 1.4 ml oftriethylamine. To the solution was added 1.4 ml of isobutylchloroformate with stirring and cooling in an ice-salt bath. Afteradditional stirring for 15 minutes, 0.87 gram of N-methyl-n-butylaminewas added to the mixture. Then the reaction mixture was stirredcontinuously for 40 minutes at room temperature. The solvent was removedby distillation under reduced pressure, below 40° C. The residue wasextracted with 100 ml of ethyl acetate and the extract was washedsuccessively with a 10% aqueous citric acid solution, saturated sodiumchloride solution, saturated aqueous sodium bicarbonate solution, andfinally with saturated aqueous sodium chloride solution. The organiclayer was dried over anhydrous Na₂ SO₄, filtered and concentrated togive N^(G) -nitro-N²-(tert-butyloxy-carbonyl)-N-(n-butyl)-N-methyl-L-argininamide. Thismaterial was added to ethyl acetate containing 10% dry HCl and allowedto stand for 2 hours, and N^(G)-nitro-N-(n-butyl)-N-methyl-L-argininamide hydrochloride wasprecipitated.

To a mixture of 3.0 gram of N^(G)-nitro-N-(n-butyl)-N-methyl-L-argininamide hydrochloride, 30 ml ofdichloromethane and 4.1 gram of triethylamine was added 3.0 gram ofdansyl chloride with stirring and cooling in an ice-bath. After stirringat 0° C. for 24 hours, 20 ml of water was added to the reaction mixture.After separation of the aqueous layer, the dichloromethane layer wasdried over anhydrous Na₂ SO₄. The Na₂ SO₄ was removed by filtration andthe solution was evaporated to give a viscous oily product. The productwas purified by chromatography using chloroform containing 10% methanolas the elutant and silica gel as the carrier. Powdery N^(G) -nitro-N²-dansyl-N-(n-butyl)-N-methyl-L-argininamide was obtained in 73% yield(based on N^(G) -nitro-N-(n-butyl)-N-methyl-L-argininamidehydrochloride).

    ______________________________________                                        Elemental analysis (as C.sub.23 H.sub.35 O.sub.5 N.sub.7 S)                            C           H      N                                                 ______________________________________                                        Calculated:                                                                              52.96         6.76   18.48                                         Found:     53.12         7.09   18.27                                         ______________________________________                                    

A 1.0 gram amount of N^(G) -nitro-N²-dansyl-N-(n-butyl)-N-methyl-L-argininamide was dissolved in 20 ml ofethanol and 5 ml of acetic acid. A 50 mg amount of palladium catalystwas added and the mixture was shaken in a stream of hydrogen for 100hours at room temperature. After filtering off the catalyst, thefiltrate was evaporated to give a viscous oily product. Reprecipitationfrom methanol-ethyl ether gave N²-dansyl-N-(n-butyl)-N-methyl-L-argininamide diacetate in powder form in83% yield.

    ______________________________________                                        Elemental analysis (as C.sub.23 H.sub.36 O.sub.3 N.sub.6 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.35         7.43   14.09                                         Found:     54.33         7.43   14.00                                         ______________________________________                                    

EXAMPLE 27

A 1.0 gram amount of N^(G) -nitro-N² -dansyl-N,N-diethyl-L-argininamide,which was prepared in the same manner as described in Example 26, wasdissolved in a mixture of 25 ml of ethanol and 5 ml of acetic acid.

A 50 mg amount of palladium black was added and the mixture was shakenin a stream of hydrogen for 50 hours at 30° C. After removal of thecatalyst, the solvent was evaporated to obtain a viscous oily residue.Reprecipitation from methanol-ethyl ether gave N²-dansyl-N,N-diethyl-L-argininamide diacetate in powder form in 71%yield.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.34 O.sub.3 N.sub.6 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              53.59         7.27   14.42                                         Found:     53.99         7.30   14.82                                         ______________________________________                                    

EXAMPLE 28

A 5.4 gram amount of N^(G),N^(G) -dibenzyloxycarbonyl-N²-(tertbutyloxy-carbonyl)-L-arginine was dissolved in a mixture of 50 mlof dry tetrahydrofuran and 1.4 ml of triethylamine. To the solution wasadded 1.4 ml of isobutyl chloroformate with stirring and cooling in anice-salt bath. After additional stirring for 15 minutes, 1.17 gram ofβ-alaine ethyl ester was added to the mixture, and the reaction mixturewas stirred further for 40 minutes at room temperature. The solvent wasremoved by distillation under reduced pressure, below 40° C. The residuewas extracted with 100 ml of ethyl acetate and the extract was washedsuccessively with 10% aqueous citric acid solution, saturated sodiumchloride solution, saturated aqueous sodium bicarbonate solution, andfinally with saturated aqueous sodium chloride solution. The organiclayer was dried over anhydrous Na₂ SO₄, filtered and concentrated togive N^(G), N^(G) -dibenzyloxycarbonyl-N²-(tert-butyloxycarbonyl)-N-(2-ethoxycarbonylethyl)-L-arginamide.

The material was added to 30 ml of formic acid and was allowed to standovernight. The formic acid was removed by distillation under reducedpressure, and the residue was washed with ethyl ether. Thus N^(G), N^(G)-dibenzyloxycarbonyl-N-(2-ethoxycarbonylethyl)-L-argininamide formatewas obtained.

To a mixture of 2.7 gram of the thus obtained N^(G),N^(G)-dibenzyloxycarbonyl-N-(2-ethoxycarbonylethyl)-L-argininamide formate,30 ml of dichloromethane and 0.6 g of triethylamine was added 1.6 g ofdansyl chloride with stirring and cooling in an ice-bath. After stirringat 0° C. for 2 hours, 20 ml of water was added to the reaction mixture.After separation of the aqueous layer, the dichloromethane solution wasdried over anhydrous Na₂ SO₄. The Na₂ SO₄ was removed by filtration andthe solvent was removed by distillation to give a viscous oily product.The product was washed well with ethyl ether and purified byreprecipitation from a dichloromethane-petroleum ether mixture.N^(G),N^(G) -dibenzyloxycarbonyl-N²-dansyl-N-(2-ethoxycarbonyl-ethyl)-L-argininamide was obtained in powderform in 92% yield (based don N^(G),N^(G)-dibenzyloxycarbonyl-N-(2-ethoxycarbonyl-ethyl)-L-argininamide formate).

    ______________________________________                                        Elemental analysis (as C.sub.39 H.sub.46 N.sub.6 O.sub.9 S)                            C           H      N                                                 ______________________________________                                        Calculated:                                                                              60.45         5.98   10.85                                         Found:     60.09         5.69   10.78                                         ______________________________________                                    

A 0.77 g amount of the thus obtained N^(G), N^(G)-dibenzyloxycarbonyl-N² -dansyl-N-(2-ethoxycarbonylethyl)-L-argininamidewas dissolved in a mixture of 50 ml of ethanol and 5 ml of acetic acid.A 50 mg amount of palladium black was added to the mixture, which wasthen shaken in a stream of hydrogen for 48 hours at room temperature.After filtering off the catalyst, the filtrate was evaporated to give aviscous oily product. The product was purified as in Example 31 and N²-dansyl-N-(2-ethoxycarbonylethyl)-L-argininamide diacetate wasquantitatively obtained.

    ______________________________________                                        Elemental analysis (as C.sub.23 H.sub.34 O.sub.4 N.sub.6 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              51.75         6.76   13.41                                         Found:     51.58         6.83   13.56                                         ______________________________________                                    

EXAMPLE 29

A 1.0 gram amount of N^(G) -nitro-N² -dansyl-N-(n-butyl)-L-arginiamide,which was preparred in the same manner as described in Example 26, wasdissolved in a mixture of 1 ml of anisole and 2 ml of hydrogen fluoride,and the mixture was stirred for 30 minutes in an ice-bath. The hydrogenfluoride was evaporated in vacuo to afford an oily product, which waswashed well with 100 ml of dry ethyl ether to remove the hydrogenfluoride. The thus obtained powdery product was neutralized with asolution of 3 ml of triethylamine in a small amount of water. Then N²-dansyl-N-(n-butyl)-L-argininamide monohydrate was obtained in 73%yield; mp. 145°-148° C.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.34 O.sub.3 N.sub.6 S.H.sub.2 O)                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.98         7.55   17.49                                         Found:     54.87         7.38   17.21                                         ______________________________________                                    

EXAMPLE 30

To 3.0 gram of N^(G) -nitro-N²-dansyl-N-(2-phenylcarbamoylethyl)-L-argininamide, which was prepared inthe same manner as described in Example 26, was added 3 ml of hydrogenfluoride at -80° C., and the mixture was stirred for 30 minutes in anice-bath. The hydrogen fluoride was evaporated in vacuo to afford anoily product, which was washed well with 100 ml of dry ethyl ether toremove the hydrogen fluoride. The thus obtained oily product wasdissolved in a small amount of alcohol and neutralized with 2 ml oftriethylamine. The residue obtained by distilling off the alcohol waswashed with water, and dissolved in ethyl acetate. The solution wasdried over anhydrous Na₂ SO₄, and the residue obtained by distilling offthe ethyl acetate was dissolved in a small amount of acetic acid. Theresidue obtained after distilling the acetic acid was washed with dryethyl ether, and N² -dansyl-N-(2-phenylcarbamoylethyl)-L-argininamidediacetate was obtained in powder form in 77% yield.

    ______________________________________                                        Elemental analysis (as C.sub.27 H.sub.35 O.sub.4 N.sub.7 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              55.26         6.43   14.56                                         Found:     54.98         6.26   14.39                                         ______________________________________                                    

EXAMPLE 31

A 2.68 gram amount of N^(G) -nitro-N²-dansyl-N-(2-ethoxycarbonylmethyl)-L-argininamide, which was prepared inthe same manner as described in Example 26, was dissolved in a mixtureof 50 ml of ethanol and 5 ml of acetic acid.

A 50 mg amount of palladium black was added and the mixture was shakenin a stream of hydrogen for 100 hours at room temperature. After removalof the catalyst, the solvent was evaporated to obtain a viscous oilyresidue. Reprecipitation from ethanol-ethyl ether quantitatively gave N²-dansyl-N-(2-ethoxycarbonylmethyl)-L-argininamide diacetate in powderform.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.32 C.sub.5 N.sub.6 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              50.97         6.58   13.72                                         Found:     50.68         6.35   14.01                                         ______________________________________                                    

EXAMPLE 32

A 1.0 gram amount of N^(G),N^(G) -dibenzyloxycarbonyl-N²-dansyl-N-methoxyethyl-L-argininamide, which was prepared in the samemanner as described in Example 28, was dissolved in a mixture of 25 mlof ethanol and 5 ml of acetic acid.

A 50 mg amount of 10% palladium black was added and the mixture wasshaken in a stream of hydrogen for 10 hours at room temperature. Afterremoval of the catalyst, the solvent was evaporated to obtain a viscousoily residue. To the residue about 15 ml of water and 3 ml oftriethylamine were added, and the mixture was allowed to stand undercooling. N² -dansyl-N-methoxyethyl-L-argininamide dihydrate was obtainedin 93% yield; m.p. 130°-135° C.

    ______________________________________                                        Elemental analysis (as C.sub.21 H.sub.32 O.sub.7 N.sub.6 S.2H.sub.2 O)                 C           H      N                                                 ______________________________________                                        Calculated:                                                                              50.38         7.25   16.79                                         Found:     50.69         6.95   16.99                                         ______________________________________                                    

EXAMPLE 33

A 3.0 gram amount of 1-(N^(G) -nitro-N² -dansyl-L-arginyl)piperidine,which was prepared in the same manner as described in Example 26, wasdissolved in a mixture of 50 ml of ethanol and 5 ml of acetic acid.

A 50 mg amount of palladium black was added and the mixture was shakenin a stream of hydrogen for 120 hours at room temperature. After removalof the catalyst, the solvent was evaporated to give a viscous oilyresidue.

Reprecipitation from methanol-ethyl ether gave 1-(N²-dansyl-L-arginyl)piperidine diacetate in powder form in 70% yield.

    ______________________________________                                        Elemental analysis (as C.sub.23 H.sub.34 O.sub.3 N.sub.6 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              54.53         7.12   14.13                                         Found:     54.31         7.11   14.13                                         ______________________________________                                    

EXAMPLE 34

A 3.0 gram amount of 4-methyl-1-(N^(G),N^(G) -dibenzyloxycarbonyl-N²-dansyl-L-arginyl)piperidine, which was prepared in the same manner asdescribed in Example 28, was dissolved in a mixture of 50 ml of ethanoland 5 ml of acetic acid.

A 50 mg amount of palladium black was added and the mixture was shakenin a stream of hydrogen for 10 hours at room temperature. After removalof the catalyst, the solvent was evaporated to give a viscous oilyresidue.

Reprecipitation from methanol-ethyl ether gave 4-methyl-1-(N²-dansyl-L-arginyl)piperidine diacetate in powder form in 80% yield.

    ______________________________________                                        Elemental analysis (as C.sub.24 H.sub.36 O.sub.3 N.sub.6 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              55.24         7.29   13.81                                         Found:     55.01         7.49   14.00                                         ______________________________________                                    

EXAMPLE 35

To 2.0 grams of 1-(N^(G) -nitro-N² -dansyl-L-arginyl)pyrrolidine, whichwas prepared in the same manner as described in Example 26, was added 2ml of hydrogen fluoride at -80° C., and the mixture was stirred for 1hour in an ice-bath. The hydrogen fluoride was evaporated in vacuo toafford an oily product, which was washed well with 100 ml of dry ethylether to remove the hydrogen fluoride. The ether was removed bydecantation and the obtained oily product was dissolved in a smallamount of water and neutralized with triethylamine. An oily productdeposited which was collected and dissolved in ethanol containing 10%acetic acid. Ethyl ether was added to the solution to reprecipitate theproduct, 1-(N² -dansyl-L-arginyl)pyrrolidine diacetate, in 65% yield.

    ______________________________________                                        Elemental analysis (as C.sub.22 H.sub.32 O.sub.3 N.sub.6 S.2CH.sub.3          COOH)                                                                                  C           H      N                                                 ______________________________________                                        Calculated:                                                                              53.77         6.94   14.47                                         Found:     52.58         7.16   14.21                                         ______________________________________                                    

EXAMPLE 36

A 0.5 g amount of 4 acetyl-1-(N^(G) -nitro-N²-dansyl-L-arginyl)piperidine, which was prepared in the same manner asdescribed in Example 26, was dissolved in a mixture of 0.39 g of anisoleand 2 ml of hydrogen fluoride at -80° C., and the mixture was stirredfor 30 minutes in an ice-bath. The hydrogen fluoride was evaporated invacuo to afford an oily product, which was washed well with 100 ml ofdry ethyl ether to remove the hydrogen fluoride. The ether layer wasremoved by decantation, and the thus obtained oily product was dissolvedin methanol. To the solution was added ethyl ether to reprecipitate theproduct, 4-acetyl-1-(N² -dansyl-L-arginyl)piperidine dihydrofluoride, in72% yield.

    ______________________________________                                        Elemental analysis (as C.sub.25 H.sub.36 O.sub.4 N.sub.6 S.2HF)                        C           H      N                                                 ______________________________________                                        Calculated:                                                                              53.94         6.88   15.10                                         Found:     53.80         6.80   14.92                                         ______________________________________                                    

Various other N² -dansyl-L-argininamides or acid addition salts thereofwere synthesized in accordance with the procedures of the aboveexamples, and the test results are summarized in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Compound                                                                       ##STR25##                        time by a factorcoagulationprolong                                           therequired toConcentration                                                            processPreparation                                                                        Lower: FoundUpper:                                                           CalculatedElemental                                                           analysis                 Sample No.                                                                          R                  Addition moiety                                                                       of 2 (μM)                                                                          (Ex. No.)                                                                           m.p. (°C.)                                                                   C  H  N                  __________________________________________________________________________    1     OCH.sub.3          2HCl.H.sub.2 O                                                                        30      1 and 12                                                                            147-150                                                                             44.62                                                                            6.11                                                                             13.69                                                                   44.42                                                                            6.17                                                                             13.64              2     OC.sub.2 H.sub.5   2HCl    8       2     140-144                                                                             47.24                                                                            6.15                                                                             13.77                                                                   46.95                                                                            6.21                                                                             14.60              3     On-C.sub.3 H.sub.7 2HCl    2       12    ≧120                                                                         48.27                                                                            6.37                                                                             13.40                                                                   48.50                                                                            6.21                                                                             13.18              4     OCH(CH.sub.3).sub.2                                                                              2HCl    30      5     110-120                                                                             48.27                                                                            6.37                                                                             13.40                                                                   47.90                                                                            6.08                                                                             13.21              5     On-C.sub.4 H.sub.9 2TsOH   2       7 and 13                                                                            160-164                                                                             53.51                                                                            6.11                                                                             8.67                                                                    53.32                                                                            6.14                                                                             8.93               6     OCH.sub.2 CH(CH.sub.3).sub.2                                                                     2TsOH   110     9     146-151                                                                             53.51                                                                            6.11                                                                             8.67                                                                    53.54                                                                            6.11                                                                             8.65               7     On-C.sub.5 H.sub.11                                                                              2TsOH   5       8     164-169                                                                             54.06                                                                            6.25                                                                             8.52                                                                    53.86                                                                            6.10                                                                             8.53               8     OCH.sub.2 CH.sub.2 CH(CH.sub.3).sub.2                                                            2TsOH   10      10    163-168                                                                             54.06                                                                            6.25                                                                             8.52                                                                    53.74                                                                            6.09                                                                             8.57               9     On-C.sub.6 H.sub.13                                                                              2TsOH   8       6     190-193                                                                             54.59                                                                            6.39                                                                             8.38                                                                    54.33                                                                            6.48                                                                             8.11               10                                                                                   ##STR26##         2TsOH   50      4     170-174                                                                             55.60 55.37                                                                      6.65 6.59                                                                        8.11 8.18          11                                                                                   ##STR27##         2TsOH   2       3     150-153                                                                             55.63 55.33                                                                      5.63 5.61                                                                        8.32 8.19          12    OCH.sub.2 CHCHCH.sub.3                                                                           2TsOH   2       11    148-153                                                                             53.64                                                                            5.88                                                                             8.69                                                                    53.64                                                                            5.90                                                                             8.35               13    OCH.sub.2 CH.sub.2 CCH                                                                           2TsOH   4       11    133-143                                                                             53.78                                                                            5.64                                                                             8.71                                                                    53.51                                                                            5.52                                                                             8.78               14                                                                                   ##STR28##         2TsOH   60      11    177-182                                                                             54.72 54.33                                                                      6.16 6.18                                                                        8.40 8.17          15                                                                                   ##STR29##         2TsOH   20      11    144-150                                                                             53.15 52.57                                                                      5.91 5.89                                                                        8.38 8.30          16    OCH.sub.2 CH.sub.2 OCH.sub.3                                                                     2TsOH   5       11    171.5-173                                                                           51.90                                                                            5.85                                                                             8.65                                                                    51.40                                                                            5.77                                                                             8.25               17    OCH.sub.2 CH.sub.2 CH.sub.2 Cl                                                                   2TsOH   4       11    140-145                                                                             50.74                                                                            5.60                                                                             8.45                                                                    50.30                                                                            5.50                                                                             8.01               18    OCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 Cl                                                          2TsOH   7       11    177-194                                                                             51.75                                                                            5.70                                                                             8.56                                                                    51.95                                                                            5.81                                                                             8.47               19                                                                                   ##STR30##         2TsOH   200     11    155-163                                                                             50.69 51.04                                                                      5.67 5.85                                                                        9.85 9.58          20                                                                                   ##STR31##         H.sub.2 O                                                                             100     18    220-222                                                                             53.08 53.34                                                                      7.13 7.18                                                                        18.57 18.86        21                                                                                   ##STR32##         H.sub.2 O                                                                             15      15    146-150                                                                             54.06 53.82                                                                      7.35 7.45                                                                        18.02 18.13        22                                                                                   ##STR33##         H.sub.2 O                                                                             25      14 and 29                                                                           145-148                                                                             54.98 54.72                                                                      7.55 7.61                                                                        17.49 17.25        23                                                                                   ##STR34##         H.sub.2 O                                                                             125     29    140-143                                                                             55.85 56.10                                                                      7.74 7.52                                                                        16.99 17.29        24                                                                                   ##STR35##         H.sub.2 O                                                                             20      19    130-135                                                                             56.67 56.38                                                                      7.93 7.59                                                                        16.53 16.34        25                                                                                   ##STR36##         H.sub.2 O                                                                             100     20    240-243                                                                             59.50 59.50                                                                      7.99 7.90                                                                        16.66 16.68        26                                                                                   ##STR37##         --      1000    16    218-221                                                                             56.23 56.38                                                                      7.19 7.20                                                                        18.74 18.94        27                                                                                   ##STR38##         --      100     21    155-160                                                                             57.12 56.82                                                                      7.41 7.41                                                                        18.17 17.90        28                                                                                   ##STR39##         2H.sub.2 O                                                                            4       22, 24 and 32                                                                       130-135                                                                             50.38 50.03                                                                      7.25 6.95                                                                        16.79 16.99        29                                                                                   ##STR40##         --      65      32    powder                                                                              55.32 55.00                                                                      6.96 7.10                                                                        17.60 17.96        30                                                                                   ##STR41##         --      5       26    230-232                                                                             55.32 55.34                                                                      6.96 7.16                                                                        17.60 17.69        31                                                                                   ##STR42##         2CH.sub.3 CO.sub.2 H                                                                  160     31    powder                                                                              50.97 50.68                                                                      6.58 6.35                                                                        13.72 14.01        32                                                                                   ##STR43##         2HCl    22      31    powder                                                                              46.72 46.58                                                                      5.70 5.91                                                                        14.86 14.97        33                                                                                   ##STR44##         2CH.sub.3 CO.sub.2 H                                                                  5       28    powder                                                                              51.75 51.58                                                                      6.76 6.83                                                                        13.41 13.56        34                                                                                   ##STR45##         2CH.sub.3 CO.sub.2 H                                                                  500     31    powder                                                                              52.49 52.51                                                                      6.92 7.13                                                                        13.12 13.01        35                                                                                   ##STR46##         H.sub.2 O                                                                             10      32    140-145                                                                             54.29 53.92                                                                      6.94 6.88                                                                        18.09 18.09        36                                                                                   ##STR47##         2H.sub.2 O                                                                            200     32    165-168                                                                             52.26 52.00                                                                      7.10 7.10                                                                        17.42 17.53        37                                                                                   ##STR48##         --      40      23    253-256                                                                             59.73 59.96                                                                      7.62 7.48                                                                        16.72 16.73        38                                                                                   ##STR49##         2CH.sub.3 CO.sub.2 H                                                                  100     27    powder                                                                              55.80 55.90                                                                      6.36 6.48                                                                        13.95 14.15        39                                                                                   ##STR50##         --      50      29    244-246                                                                             60.46 59.97                                                                      6.50 6.46                                                                        16.92 16.77        40                                                                                   ##STR51##         2H.sub.2 O                                                                            150     17    143-147                                                                             57.13 57.34                                                                      7.01 6.72                                                                        15.38 15.64        41                                                                                   ##STR52##         H.sub.2 O                                                                             27      26    131-135                                                                             59.75 59.39                                                                      7.06 6.98                                                                        15.49 15.23        42                                                                                   ##STR53##         2CH.sub.3 CO.sub.2 H                                                                  >500    30    powder                                                                              55.26 54.98                                                                      6.43 6.26                                                                        14.56 14.39        43                                                                                   ##STR54##         2CH.sub.3 CO.sub.2 H                                                                  370     31    powder                                                                              52.73 52.54                                                                      6.80 6.63                                                                        15.38 15.13        44                                                                                   ##STR55##         2HF             36    powder                                                                              52.26 52.02                                                                      6.48 6.48                                                                        15.90 15.81        45                                                                                   ##STR56##         2CH.sub.3 CO.sub.2 H                                                                  55      27    powder                                                                              53.59 53.99                                                                      7.27 7.30                                                                        14.42 14.82        46                                                                                   ##STR57##         2CH.sub.3 CO.sub.2 H                                                                  39      27    powder                                                                              56.41 55.99                                                                      7.89 7.65                                                                        13.16 13.36        47                                                                                   ##STR58##         2CH.sub.3 CO.sub.2 H                                                                  2       26    powder                                                                              54.35 54.33                                                                      7.43 7.43                                                                        14.09 14.00        48                                                                                   ##STR59##         2CH.sub.3 CO.sub.2 H                                                                  50      27    powder                                                                              55.06 54.99                                                                      7.59 7.89                                                                        13.76 14.01        49                                                                                   ##STR60##         2CH.sub.3 CO.sub.2 H                                                                  59      31    powder                                                                              51.75 51.49                                                                      6.76 6.84                                                                        13.41 13.06        50                                                                                   ##STR61##         2CH.sub.3 CO.sub.2 H                                                                  2.5     31    powder                                                                              51.75 51.49                                                                      6.76 6.93                                                                        13.41 13.70        51                                                                                   ##STR62##         2CH.sub.3 CO.sub.2 H                                                                  15      31    powder                                                                              52.49 52.66                                                                      6.92 7.18                                                                        13.12 13.51        52                                                                                   ##STR63##         2CH.sub.3 CO.sub.2 H                                                                  7       27    powder                                                                              57.12 56.92                                                                      6.71 6.79                                                                        13.33 13.61        53                                                                                   ##STR64##         2CH.sub.3 CO.sub.2 H                                                                  3.3     35    powder                                                                              53.77 53.58                                                                      6.94 7.16                                                                        14.47 14.21        54                                                                                   ##STR65##         2CH.sub.3 CO.sub.2 H                                                                  780     31    powder                                                                              53.36 53.39                                                                      6.79 7.05                                                                        12.88 12.70        55                                                                                   ##STR66##         2CH.sub.3 CO.sub.2 H                                                                  0.9     25 and 33                                                                           powder                                                                              54.53 54.23                                                                      7.12 7.11                                                                        14.13 14.43        56                                                                                   ##STR67##         2CH.sub.3 CO.sub.2 H                                                                  1.3     33    powder                                                                              55.24 55.01                                                                      7.29 7.50                                                                        13.81 14.09        57                                                                                   ##STR68##         2CH.sub.3 CO.sub.2 H                                                                  6.5     33    powder                                                                              55.24 55.13                                                                      7.29 7.44                                                                        13.81 14.00        58                                                                                   ##STR69##         2CH.sub.3 CO.sub.2 H                                                                  0.3     34    powder                                                                              55.24 55.01                                                                      7.29 7.49                                                                        13.81 14.00        59                                                                                   ##STR70##         2CH.sub.3 CO.sub.2 H                                                                  0.1     33    powder                                                                              55.93 55.87                                                                      7.45 7.66                                                                        13.50 13.81        60                                                                                   ##STR71##         2CH.sub.3 CO.sub.2 H                                                                  1.0     33    powder                                                                              56.78 56.61                                                                      7.60 8.00                                                                        13.20 13.06        61                                                                                   ##STR72##         2CH.sub.3 CO.sub.2 H                                                                  1.0     33    powder                                                                              56.78 56.48                                                                      7.60 7.64                                                                        13.20 13.10        62                                                                                   ##STR73##         2CH.sub.3 CO.sub.2 H                                                                  2.8     31    powder                                                                              53.36 53.69                                                                      6.80 7.18                                                                        12.88 12.91        63                                                                                   ##STR74##         2CH.sub.3 CO.sub.2 H                                                                  100     31    powder                                                                              53.92 53.68                                                                      6.95 6.76                                                                        13.48 13.35        64                                                                                   ##STR75##         2CH.sub.3 CO.sub.2 H                                                                  0.9     33    powder                                                                              55.24 55.49                                                                      7.29 7.38                                                                        13.81  14.11       65                                                                                   ##STR76##         2CH.sub.3 CO.sub.2 H                                                                  1       33    powder                                                                              55.93 55.71                                                                      7.45 7.73                                                                        13.50 13.20        66                                                                                   ##STR77##         2CH.sub.3 CO.sub.2 H                                                                  2       33    powder                                                                              56.78 56.59                                                                      7.60 7.87                                                                        13.20 13.00        67                                                                                   ##STR78##         2CH.sub.3 CO.sub.2 H                                                                  1.5     33    powder                                                                              52.33 52.53                                                                      6.76 7.00                                                                        14.09 14.39        68                                                                                   ##STR79##         2CH.sub.3 CO.sub.2 H                                                                  20      33    powder                                                                              53.83 53.55                                                                      7.10 7.17                                                                        13.45 13.81        69                                                                                   ##STR80##         2CH.sub.3 CO.sub.2 H                                                                  10      33    powder                                                                              56.76 56.62                                                                      7.30 7.59                                                                        13.24 13.52        70                                                                                   ##STR81##         2CH.sub.3 CO.sub.2 H                                                                  0.67    33    powder                                                                              57.31  57.03                                                                     6.41 6.57                                                                        13.37 13.62        71                                                                                   ##STR82##         2CH.sub.3 CO.sub.2 H                                                                  2       33    powder                                                                              53.18 53.01                                                                      7.11 7.00                                                                        16.08 15.79        72                                                                                   ##STR83##         2CH.sub.3 CO.sub.2 H                                                                  6.5     33    powder                                                                              57.92 57.71                                                                      6.59 6.58                                                                        13.08 12.97        73                                                                                   ##STR84##         2HF             36    powder                                                                              52.26 52.02                                                                      6.48 6.48                                                                        15.90 15.81        __________________________________________________________________________

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and intended to be covered by Letters Patentis:
 1. N² -Dansyl-L-Argininamides having the formula ##STR85## whereinR₂ is hydrogen or methyl and R₁ is --CH₂ CO₂ C₂ H₅ or --CH₂ CH₂ CH₂ CO₂C₂ H₅.
 2. The compound of claim 1, whereinR₂ =H R₁ =--CH₂ --CO₂ C₂ H₅.3. The compound of claim 1, whereinR₂ =H R₁ =--CH₂ --CH₂ --CH₂ --CO₂--C₂ H₅.
 4. The compound of claim 1, whereinR₂ =CH₃ R₁ =--CH₂ --CO₂ C₂H₅.
 5. An antithrombotic composition which comprises anantithrombotically effective amount of a compound of claim 1 and apharmaceutically acceptable carrier.
 6. A method for inhibiting activityand suppressing activation of thrombin in vivo, which comprisesadministering to a patient a pharmaceutically effective amount of thecomposition according to claim 5.